Yeast and fungal infections represent a major cause of morbidity and mortality among immunocompromised patients. The number of immunocompromised patients at risk of yeast and fungal infection continues to increase each year, as does the spectrum of fungal and yeast agents causing disease. Mortality from fungal infections, particularly invasive fungal infections, is 30% or greater, in certain risk groups. The array of available antifungal agents is growing; however, so too is the recognition of both intrinsic and emerging resistance to antifungal drugs. These factors are contributing to the increased need for cost containment in laboratory testing and have led to laboratory consolidation in testing procedures.
Invasive fungal infections are on the increase. In 2003, it was estimated that there were 9 million at risk patients, of which 1.2 million developed infection. Candida spp. and Aspergillus spp. now rank as the most prominent pathogens infecting immunosupressed patients. In particular, infections are common in the urinary tract, the respiratory system and the bloodstream, at the site of insertion of stents, catheters and orthopaedic joints. Approximately, 10% of the known Candida spp. have been implicated in human infection. Invasive candidiasis occurs when candida enters the bloodstream and it is estimated to occur at a frequency of 8/100,000 population in the US with a mortality rate of 40%. Candida albicans is the 4th most common cause of bloodstream infection. Aspergillosis usually begins as a pulmonary infection that can progress to a life-threatening invasive infection in some patients and has a mortality rate of greater than 90%. Emerging mycoses agents include Fusarium, Scedosporium, Zygomycetes and Trichosporon spp. (“Stakeholder Insight: Invasive fungal infections”, Datamonitor, January 2004).
Immunocompromised patients, including transplant and surgical patients, neonates, cancer patients, diabetics and those with HIV/AIDs are at high risk of developing invasive fungal infections (Datamonitor report: Stakeholder opinion—Invasive fungal infections, options outweigh replacements 2004). A large number of severe cases of sepsis are reported each year. Despite improvements in its medical management, sepsis still constitutes one of the greatest challenges in intensive care medicine. Microorganisms (bacteria, fungi and yeast) responsible for causing sepsis are traditionally detected in hospital laboratories with the aid of microbiological culture methods with poor sensitivity (25-82%), which are very time-consuming, generally taking from two to five days to complete, and up to eight days for the diagnosis of fungal infections. Definitive diagnosis of infections caused by yeast or fungi is usually based on either, the recovery and identification of a specific agent from clinical specimens or microscopic demonstration of fungi with distinct morphological features. However, there are numerous cases where these methods fail to provide conclusive proof as to the infecting agent. In these instances, the detection of specific host antibody responses can be used, although again this can be affected by the immune status of the patient. Time is critical in the detection and identification of bloodstream infections typically caused by bacteria, yeasts or fungi. Effective treatment depends on finding the source of infection and making appropriate decisions about antibiotics or antifungals quickly and efficiently. Only after pathogens are correctly identified can targeted therapy using a specific antibiotic or antifungal begin. Many physicians would like to see the development of better in vitro amplification and direct detection diagnostic techniques for the early diagnosis of yeast and fungi (“Stakeholder Insight: Invasive fungal infections”, Datamonitor, January 2004). Recently Roche™ launched a real time PCR based assay (Septifast™), for the detection of bacterial, fungal and yeast DNA in clinical samples. Therefore, there is a clear need for the development of novel rapid diagnostic tests for clinically significant bacterial and fungal pathogens for bioanalysis applications in the clinical sector. This has led the current inventors to identify novel fungal and yeast nucleic acid targets for application in Nucleic Acid Diagnostics (NAD) tests.
Fungal and yeast nucleic acid based diagnostics have focused heavily on the ribosomal RNA (rRNA) genes, RNA transcripts, and their associated DNA/RNA regions. The rRNA genes are highly conserved in all fungal species and they also contain divergent and distinctive intergenic transcribed spacer regions. Ribosomal rRNA comprises three genes: the large sub-unit gene (28S), the small sub-unit gene (18S) and the 5.8S gene. The 28S and 18S rRNA genes are separated by the 5.8S rRNA and two internal transcribed spacers (ITS1 and ITS2). Because the ITS region contains a high number of sequence polymorphisms, numerous researchers have concentrated their efforts on these as targets (Atkins and Clark, 2004). rRNA genes are also multicopy genes with >10 copies within the fungal genome.
A number of groups are working on developing new assays for fungal and yeast infections. US2004044193 relates to, amongst a number of other aspects, the transcription factor CaTEC1 of Candida albicans; inhibitors thereof, and methods for the diagnosis and therapy of diseases which are connected with a Candida infection; and also diagnostic and pharmaceutical compositions which contain the nucleotide sequences, proteins, host cells and/or antibodies. WO0183824 relates to hybridization assay probes and accessory oligonucleotides for detecting ribosomal nucleic acids from Candida albicans and/or Candida dubliniensis. U.S. Pat. No. 6,017,699 and U.S. Pat. No. 5,426,026 relate to a set of DNA primers, which can be used to amplify and speciate DNA from five medically important Candida species. U.S. Pat. No. 6,747,137 discloses sequences useful for diagnosis of Candida infections. EP 0422872 and U.S. Pat. No. 5,658,726 disclose probes based on 18S rRNA genes, and U.S. Pat. No. 5,958,693 discloses probes based on 28S rRNA, for diagnosis of a range of yeast and fungal species. U.S. Pat. No. 6,017,366 describes sequences based on chitin synthase gene for use in nucleic acid based diagnostics for a range of Candida species.
It is clear though, that development of faster, more accurate diagnostic methods are required, particularly in light of the selection pressure caused by modern antimicrobial treatments which give rise to increased populations of resistant virulent strains with mutated genome sequences. Methods that enable early diagnosis of microbial causes of infection enable the selection of a specific narrow spectrum antibiotic or antifungal to treat the infection (Datamonitor report: Stakeholder opinion—Invasive fungal infections, options outweigh replacements 2004; Datamonitor report: Stakeholder Opinion-Sepsis, under reaction to an overreaction, 2006). Eukaryotic initiation factor 2 (eIF2) is a heterotrimer composed of three subunits eIF2alpha (eIF2α), eIF2beta (eIF2β) and eIF2gamma (eIF2γ). eIF2 is the eukaryotic translation initiation factor 2, which is a heterotrimeric G-protein required for GTP-dependent delivery of initiator tRNA to the ribosome. The eIF2 gamma subunit (eif2γ) has a similar amino acid sequence to prokaryotic translation elongation factor EF-Tu which was patented as molecular diagnostics targets for micro-organisms (Alone and Dever, 2006; Dorris et al., 1995; Erickson et al., 1996 and 1997).
There are currently 171 sequences of eIF2γ available in NCBI GenBank database including 3 Candida spp. including 2 annotated eIF2γ sequences for C. albicans and one hypothetical protein for C. glabrata with 78% homology to C. albicans eIF2γ and 6 Aspergillus spp. sequences, 3 annotated as eIF2γ and 3 hypothetical sequences. The published sequences are approximately 1600 base pairs in length providing a number of sequence regions that are suitable for PCR primer and probe design for species identification of Candida and Aspergillus spp. For Candida, the inventors focussed on the region of the eIF2 gamma gene equivalent to base pair (bp) position 718 to by position 1040 in C. albicans. For Aspergillus, the inventors designed primers to amplify the region of the eIF2γ gene equivalent to by position 121 to by position 374 in Aspergillus fumigatus. 